Estradiol 3 assay

Clotted blood was collected using clot blood tube and sent immediately to the HIV‐NAT laboratory for serum separation. Blood samples were centrifuged at 1800 g (3000 rpm) for 10 minutes at 20°C. Serum was separated and transferred to labelled polypropylene tubes and stored at −20°C until analysis (less than six months). E2 was performed by Elecsys Estradiol III Assay (Roche Diagnostics GmbH, Mannheim, Germany) based on the electrocheminescence immunoassay technique. The limits of lower and upper detection were 5 and 3000 pg/mL, respectively (18.4‐11,010 pmol/L).
In addition to intensive PK analysis of TFV and E2, C₂₄ of testosterone was measured at week 3 and week 5 to indirectly assess DDI between PrEP and FHT by comparing the anti‐androgen effect of CPA before and after the presence of PrEP.

Serum and urine creatinine and magnesium, and whole blood CsA levels were analyzed with the Dimension Vista system (Siemens Healthcare Diagnostics, Deerfield, MA, USA) using an ECREA, Mg, or CsA flex^® reagent cartridge, respectively. FE Mg²⁺ was calculated using the following equation: FE_Mg = 100 × (U_Mg × S_Cr)/[(0.8 × S_Mg) × U_Cr], with U_Mg urinary excretion of Mg²⁺ (mg/dL), S_Cr serum creatinine (mg/dL), S_Mg serum Mg²⁺ (mg/dL), and U_Cr urinary excretion of creatinine (mg/dL). The serum Mg²⁺ concentration was multiplied by 0.8, since, in children, only 80% of the serum Mg²⁺ is freely filtered by the glomerulus, with the remaining part being protein-bound [28 ].
Creatinine clearance was calculated using the Bedside Swartz equation, which is the recommended equation to estimate the GFR in children [29 (link)].
Since it is known that estradiol might influence renal magnesium reabsorption [30 (link)], serum estradiol levels were measured in female children using the Elecsys Estradiol III Assay (Cobas^®, Roche Diagnostics GmbH, Mannheim, Germany). TAC levels were measured with the Elecsys Tacrolimus Assay.